3.2.1.5 Studying Cells/Microscopy/Cell Fractionation (C3) Flashcards
What you see when looking through a microscope is called the
Image
What is the biggest disadvantage of a light microscope?
- Low resolution due to ‘longer’ wavelength of light.
What are the advantages of a light microscope?
- True colour images but may sometimes require staining.
- Can observe live specimens
- Simple preparation of specimens
Define microscope resolving power.
The ability of a microscope to differentiate between 2 close together objects.
What is meant by magnification?
How much bigger an object looks under a microscope.
Magnification = Image Size ÷ Actual Size
What are the advantages of a transmission electron microscope (TEM)?
- High resolving power (0.1 nm)
- High magnification (X500, 000)
- Provides detailed images of internal structures of cells.
Name the 3 main microscopes used by scientists.
- Light microscope
- Scanning electron microscope (SEM)
- Transmission electron microscope (TEM)
What are the advantages of a scanning electron microscope?
- High resolution (20 nm)
- High magnification (X200, 000)
- 3D images of surface of the cells
Why do electron microscopes have a greater resolving power than light microscopes?
- They use electrons to interact with the specimen.
- Electrons have a shorter wavelength so interact more with the specimen.
What are the disadvantages of a transmission election microscope (TEM)?
- Special training is required before use.
- Samples must be dead as electrons are fired through a vacuum
- ‘Artefacts’ can be present in image from staining process.
- Sample must be 1 cell thick to allow electrons to penetrate specimen.
- Black and white images only so false colour must be used.
The resolving power of a light microscope is 2 µm what does this mean?
2 µm
It can differentiate between objects up to that distance apart.
What are the disadvantages of a scanning electron microscope (SEM)?
- Special training is required before use.
- Samples must be dead as electrons are fired through a vacuum and stains containing heavy elements are used.
- ‘Artefacts’ can be present in image from staining process.
- Black and white images only so false colour must be used.
- Cannot see inside specimens.
What are the main differences between scanning and transmission electron microscopes?
Does light or electons have the shortest wavelength?
Electrons
What is cell fracitonation?
The process by which cells are broken up and organelles are separated out.
Describe the stages of cell fractionation.
- Tissue is placed in a cold, buffered, isotonic solution.
- Tissue and cells are broken up using a homogeniser (blender)
- Homogenate is filtered to remove large debris.
- Nuclei in the homogenate are separated by being spun at low speed using a centrifuge (ultracentrifugation)
- Supernatent is removed leaving pellet of nuclei.
- Supernatent spun at medium speed to create pellet of mitochondrion/chloroplasts.
- Supernatent removed and spun at high speed to create pellet of ribosomes.
Before cell fractionation can take place, the tissue to be observed is placed in a cold, buffered, isotonic solution. Why is the solution cold?
To reduce enzyme activity within the cell that could break down organelles.
Before cell fractionation can take place, the tissue to be observed is placed in a cold, buffered, isotonic solution. Why is the solution isotonic?
If the solution was not of the same water potential as the tissue then organelles could burst as a result of osmotic gain or loss of water.
Before cell fractionation can take place, the tissue to be observed is placed in a cold, buffered, isotonic solution. Why is the solution buffered?
So that pH is maintained to prevent proteins denaturing
What is a homogeniser?
A blender used to break up tissues and cells and release organelles.
What is a homogenate?
The resulting fluid after homogenisation
What is a centrifuge?
A machine that spins tubes of homogenate at varying speeds (used in cell fractionation)
Name 2 organelles found in eukaryotes that cant be seen with a light microscope
mitochondria, ribosome, ER, cell surface membrane
when preparing a slide for viewing why must you press down hard on the coverslip (without breaking!)
To squash tissue to it is one cell thick so light can pass through
why should the specimen be thin?
single layer of cells
to allow light to pass through
how do you prepare a temporary mount for viewing?
add drop of water to slide
take a thin section of tissue - 1 cell thick
place on glass slide (float on water)
add stain (e.g. iodine)
place on coverslip
press down firmly
Contrast a TEM and a Optical microcope
TEM electrons - light optical
TEM greatER resolution
TEM can see smallER organelles e.g. ribosomes
TEM only dead specimens - light can view dead and live
TEM no colour - optical can
TEM needs thinnER specimen
If doing a scientific drawing at an image under the microscope what should you do to ensure the quality of it?
dont use shading
use SINGLE lines (no sketching)
add labels/annotations
dont cross label lines
add a magnification/scale bar
What type of image does an SEM provide?
3D surface
How caould you determine the mean length of a cell using an eye piece graticule?
calibrate the graticule using a ruler/stage micrometer
Measure the length of a number of cells (at random) using the graticule
calculate a mean
Give some top tips when making a biological drawing
- continuous lines - no sketching
- no shading
- draw what you see
- add a scale bar or state magnification/scale
- Add labels
Why does a sample for microscopy need to be sliced thinly?
To be a thin layer of cells, so light can pass through
In a root tip squash, what is Hydrochloric acid used for?
To kill the cells and stop mitosis
To separate the cell walls
To allow the stain to pass into the cell more easily
What does cell fractionation involve?
- break open cells and remove debris
- add cold isotonic buffer
- spin in centrifuge - most dense organelle pellets 1st
In cell fractionation which pellet would you find the chloroplasts?
second
Identify what is missing from the unit conversion diagram.
How do we calculate the magnification of an image?
Magnification = Image Size ÷ Actual Size
1 m in µm =
1 x 106 µm
What can we use to help rearrange the equations for calculating image size, actual size and magnification?
How should you calculate magnification if there is a ‘scale bar’ present on a photomicrograph?
You are given the Actual Size next to the scale bar
Measure the Image Size using a ruler
Magnification = Image Size ÷ Actual Size
1 nm in m =
1 x 109 nm
1 µm in m =
1 x 10-6 m
1 mm in m =
1 x 10-3 m
How can we calculate the size of the image being observed through microscope?
Image Size = Actual Size x Magnification
When doing cell calculation you should make sure that you are working in which units?
Micrometres
1 m in mm =
1x103 mm
How do we calculate the actual size of an image observed through a microscope?
Actual Size = Image Size ÷ Magnification
